The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

A Cyanobacterial Gene Encoding Peptidyl-Prolyl cis-trans Isomerase

The rotA gene encoding peptidyl-prolyl cis-trans isomerase has been identified, sequenced, and shown to be transcribed in the cyanobacterium Synechococcus sp. PCC7942. Inactivation of the gene by replacement of a region containing the open reading frame with a gene conferring kanamycin resistance resulted in merodiploids containing both the wild type and the modified genomic region. We were not able to isolate a kanamycin-resistant mutant in which all the genomic wild-type copies were substituted, which suggests that such replacement could have been lethal.     

Molecular evolution of the HBII-52 snoRNA cluster

HBII-52 is a human brain-specific C/D box snoRNA that potentially regulates the editing and/or alternative splicing of the serotonin receptor. Forty-two nearly identical copies of the HBII-52 gene are located immediately downstream of the SNRPN protein-coding gene in an imprinted locus associated with Prader-Willi syndrome. Other eutherian mammals, with genomic assemblies covering the corresponding locus, also have multiple orthologous copies of HBII-52. The SNRPB gene, which is known to have given rise to SNRPN through gene duplication, expresses a C/D box snoRNA, SNORD119, from its fifth intron. Here we show that, despite the fact that they lie in different positions relative to the orthologous SNRPB/SNRPN coding sequences, there are significant sequence similarities between SNORD119 and HBII-52, including the antisense element and the stem-forming regions. By analysing these snoRNAs in marsupial and eutherian mammal genomes, we reconstruct the likely evolutionary history of the HBII-52 cluster and SNORD119 and suggest that they have evolved from a common ancestor.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.     

Characterization of the human NIPSNAP1 gene from 22q12: a member of a novel gene family

Rapid progress in sequencing of human and other genomes allows high-resolution analysis of their gene content on the basis of comparison between species. We have used a combined computer and biochemical approach to characterize 135 kb of human genomic sequence from 22q12 and discovered a new 10 exon gene, termed NIPSNAP1, located between the neurofibromatosis type 2 and the pK1.3 genes. The NIPSNAP1 gene spans 26 kb of genomic sequence and shows two large introns in the 5′-region. All exon–intron junctions contain the gt/ag consensus splice site. The putative promoter of the NIPSNAP1 gene is TATA-less and resides in a GC-rich island characteristic of housekeeping genes. The NIPSNAP1 mRNA is 2.1 kb, is expressed ubiquitously at variable levels, with the highest expression in liver, is terminated by an uncommon ATTAAA polyadenylation site, and is capable of encoding a 284-amino-acid protein. This NIPSNAP1 protein has a strong sequence similarity limited to the central portion of a hypothetical protein (acc. P34492) from chromosome III of C. elegans, in which the other portions resemble a 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein. Thus, the NIPSNAP1 gene is a member of an evolutionarily well conserved, novel gene family with two members in human and mouse that have now been characterized, and one member in C. elegans. The second human gene, NIPSNAP2, is localized in the vicinity of marker D7S499 on chromosome 7. Although the function of the NIPSNAP protein family is unknown, clues about its role may reside in the co-expression of the C. elegans orthologue, within an operon encoding protein motifs known to be involved in vesicular transport.     

Microevolution of the Chromosomal Region of Acute Disease Antigen A (adaA) in the Query (Q) Fever Agent Coxiella burnetii

The acute disease antigen A (adaA) gene is believed to be associated with Coxiella burnetii strains causing acute Q fever. The detailed analysis of the adaA genomic region of 23 human- and 86 animal-derived C. burnetii isolates presented in this study reveals a much more polymorphic appearance and distribution of the adaA gene, resulting in a classification of C. burnetii strains of better differentiation than previously anticipated. Three different genomic variants of the adaA gene were identified which could be detected in isolates from acute and chronic patients, rendering the association of adaA positive strains with acute Q fever disease disputable. In addition, all adaA positive strains in humans and animals showed the occurrence of the QpH1 plasmid. All adaA positive isolates of acute human patients except one showed a distinct SNP variation at position 431, also predominant in sheep strains, which correlates well with the observation that sheep are a major source of human infection. Furthermore, the phylogenetic analysis of the adaA gene revealed three deletion events and supported the hypothesis that strain Dugway 5J108-111 might be the ancestor of all known C. burnetii strains. Based on our findings, we could confirm the QpDV group and we were able to define a new genotypic cluster. The adaA gene polymorphisms shown here improve molecular typing of Q fever, and give new insights into microevolutionary adaption processes in C. burnetii.     

Global analysis of the eukaryotic pathways and networks regulated by Salmonella typhimurium in mouse intestinal infection in vivo

Background

Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.

Results

We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.

Conclusion

We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.     

Review of microarray studies for host-intracellular pathogen interactions

Obligate intracellular bacteria are privileged soldiers on the battlefield that represent host-pathogen interactions. Microarrays are a powerful technology that can increase our knowledge about how bacteria respond to and interact with their hosts. This review summarizes the limitations inherent to host-pathogen interaction studies and essential strategies to improve microarray investigations of intracellular bacteria. We have compiled the comparative genomic and gene expression analyses of obligate intracellular bacteria currently available from microarrays. In this review we explore ways in which microarrays can be used to identify polymorphisms in different obligate intracellular bacteria such as Coxiella burnetii, Chlamydia trachomatis, Ehrlichia chaffeensis, Rickettsia prowazekii and Tropheryma whipplei. These microarray studies reveal that, while genomic content is highly conserved in obligate intracellular bacteria, genetic polymorphisms can potentially occur to increase bacterial pathogenesis. Additionally, changes in the gene expression of C. trachomatis throughout its life cycle, as well as changes in the gene expression profile of the pathogens R. prowazekii, Rickettsia rickettsii, Rickettsia typhi, T. whipplei and C. trachomatis following environmental changes, are discussed. Finally, an in vivo model of Rickettsia conorii within the skin is discussed. The gene expression analyses highlight the capacity of obligate intracellular bacteria to adapt to environmental changes and potentially to thwart the host response.     

Comprehensive Genomic Characterization of Campylobacter Genus Reveals Some Underlying Mechanisms for its Genomic Diversification

Campylobacter species.are phenotypically diverse in many aspects including host habitats and pathogenicities, which demands comprehensive characterization of the entire Campylobacter genus to study their underlying genetic diversification. Up to now, 34 Campylobacter strains have been sequenced and published in public databases, providing good opportunity to systemically analyze their genomic diversities. In this study, we first conducted genomic characterization, which includes genome-wide alignments, pan-genome analysis, and phylogenetic identification, to depict the genetic diversity of Campylobacter genus. Afterward, we improved the tetranucleotide usage pattern-based naïve Bayesian classifier to identify the abnormal composition fragments (ACFs, fragments with significantly different tetranucleotide frequency profiles from its genomic tetranucleotide frequency profiles) including horizontal gene transfers (HGTs) to explore the mechanisms for the genetic diversity of this organism. Finally, we analyzed the HGTs transferred via bacteriophage transductions. To our knowledge, this study is the first to use single nucleotide polymorphism information to construct liable microevolution phylogeny of 21 Campylobacter jejuni strains. Combined with the phylogeny of all the collected Campylobacter species based on genome-wide core gene information, comprehensive phylogenetic inference of all 34 Campylobacter organisms was determined. It was found that C. jejuni harbors a high fraction of ACFs possibly through intraspecies recombination, whereas other Campylobacter members possess numerous ACFs possibly via intragenus recombination. Furthermore, some Campylobacter strains have undergone significant ancient viral integration during their evolution process. The improved method is a powerful tool for bacterial genomic analysis. Moreover, the findings would provide useful information for future research on Campylobacter genus.     

Secondary Metabolism and Biotrophic Lifestyle in the Tomato Pathogen Cladosporium fulvum

Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mould of tomato. Analysis of its genome suggested a high potential for production of secondary metabolites (SM), which might be harmful to plants and animals. Here, we have analysed in detail the predicted SM gene clusters of C. fulvum employing phylogenetic and comparative genomic approaches. Expression of the SM core genes was measured by RT-qrtPCR and produced SMs were determined by LC-MS and NMR analyses. The genome of C. fulvum contains six gene clusters that are conserved in other fungal species, which have undergone rearrangements and gene losses associated with the presence of transposable elements. Although being a biotroph, C. fulvum has the potential to produce elsinochrome and cercosporin toxins. However, the corresponding core genes are not expressed during infection of tomato. Only two core genes, PKS6 and NPS9, show high expression in planta, but both are significantly down regulated during colonization of the mesophyll tissue. In vitro SM profiling detected only one major compound that was identified as cladofulvin. PKS6 is likely involved in the production of this pigment because it is the only core gene significantly expressed under these conditions. Cladofulvin does not cause necrosis on Solanaceae plants and does not show any antimicrobial activity. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies on C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle.     

The mouse uracil-DNA glycosylase gene: isolation of cDNA and genomic clones and mapping ung to mouse chromosome 5

Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).     

<Emphasis Type="Italic">Corynebacterium defluvii</Emphasis> sp. nov., isolated from Sewage

A Gram-positive, aerobic, non-motile, rod-shapeds, catalase-positive, and oxidase-negative strain, designated Y49^T, was isolated from sewage collected from Jilin Agricultural University, China. It grew at 20–40°C (optimum at 30°C), at pH 6.0–8.0 (optimum at 7.0) and at 0–1.0% sodium chloride (optimum at 0%). The major isoprenoid quinone was menaquinone-8 (MK-8) and the polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, four unidentified lipids, and two unidentified aminolipids. The peptidoglycan was meso-diaminopimelic acid. The cell-wall sugars were galactose, arabinose, and glucose. The fatty acids were C_9:0, C_16:0, C_16:1 ω9c, C_17:1 ω9c, C_18:3 ω6c (6,9,12), C_18:1 ω9c, and C_18:0. The DNA G+C content was 51.4 mol%. Based on the 16S rRNA gene sequence analysis, the nearest phylogenetic neighbors of strain Y49^T were Corynebacterium efficiens DSM 44549^T (97.5%), Corynebacterium callunae DSM 20147^T (97.2%), Corynebacterium deserti GIMN 1.010^T (96.8%), Corynebacterium glutamicum ATCC 13032^T (96.4%), and other species belonging to this genus (92.3–95.4%). The DNA-DNA relatedness value between strain Y49^T and C. efficiens DSM 44549^T, C. callunae DSM 20147^T, C. deserti GIMN1.010^T, and C. glutamicum ATCC 13032^T was 25.5±2.0%, 21.1±1.0%, 16.5±0.5%, and 13.5±0.9%, respectively. Based on the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain Y49^T represents a novel species of the genus Corynebacterium, for which the name Corynebacterium defluvii sp nov. is proposed. The type strain is Y49^T (= KCTC 39731^T =CGMCC 1.15506^T).     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

A Comprehensive Genetic Analysis of Candidate Genes Regulating Response to Trypanosoma congolense Infection in Mice

Background

African trypanosomes are protozoan parasites that cause “sleeping sickness” in humans and a similar disease in livestock. Trypanosomes also infect laboratory mice and three major quantitative trait loci (QTL) that regulate survival time after infection with T. congolense have been identified in two independent crosses between susceptible A/J and BALB/c mice, and the resistant C57BL/6. These were designated Tir1, Tir2 and Tir3 for Trypanosoma infection response, and range in size from 0.9–12 cM.

Principal Findings

Mapping loci regulating survival time after T. congolense infection in an additional cross revealed that susceptible C3H/HeJ mice have alleles that reduce survival time after infection at Tir1 and Tir3 QTL, but not at Tir2. Next-generation resequencing of a 6.2 Mbp region of mouse chromosome 17, which includes Tir1, identified 1,632 common single nucleotide polymorphisms (SNP) including a probably damaging non-synonymous SNP in Pram1 (PML-RAR alpha-regulated adaptor molecule 1), which was the most plausible candidate QTL gene in Tir1. Genome-wide comparative genomic hybridisation identified 12 loci with copy number variants (CNV) that correlate with differential gene expression, including Cd244 (natural killer cell receptor 2B4), which lies close to the peak of Tir3c and has gene expression that correlates with CNV and phenotype, making it a strong candidate QTL gene at this locus.

Conclusions

By systematically combining next-generation DNA capture and sequencing, array-based comparative genomic hybridisation (aCGH), gene expression data and SNP annotation we have developed a strategy that can generate a short list of polymorphisms in candidate QTL genes that can be functionally tested.